Anatomical variability, multi-modal coordinate systems, and precision targeting in the marmoset brain.

Localising accurate brain regions needs careful evaluation in each experimental species due to their individual variability. However, the function and connectivity of brain areas is commonly studied using a single-subject cranial landmark-based stereotactic atlas in animal neuroscience. Here, we address this issue in a small primate, the common marmoset, which is increasingly widely used in systems neuroscience. We developed a non-invasive multi-modal neuroimaging-based targeting pipeline, which accounts for intersubject anatomical variability in cranial and cortical landmarks in marmosets. This methodology allowed creation of multi-modal templates (MarmosetRIKEN20) including head CT and brain MR images, embedded in coordinate systems of anterior and posterior commissures (AC-PC) and CIFTI grayordinates. We found that the horizontal plane of the stereotactic coordinate was significantly rotated in pitch relative to the AC-PC coordinate system (10 degrees, frontal downwards), and had a significant bias and uncertainty due to positioning procedures. We also found that many common cranial and brain landmarks (e.g., bregma, intraparietal sulcus) vary in location across subjects and are substantial relative to average marmoset cortical area dimensions. Combining the neuroimaging-based targeting pipeline with robot-guided surgery enabled proof-of-concept targeting of deep brain structures with an accuracy of 0.2 mm. Altogether, our findings demonstrate substantial intersubject variability in marmoset brain and cranial landmarks, implying that subject-specific neuroimaging-based localization is needed for precision targeting in marmosets. The population-based templates and atlases in grayordinates, created for the first time in marmoset monkeys, should help bridging between macroscale and microscale analyses.

Discovery of dihydroquinazolinone derivatives as potent, selective, and CNS-penetrant M(1) and M(4) muscarinic acetylcholine receptors agonists.

We designed and synthesized a series of dihydroquinazolinone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Introduction of the N-carbethoxy piperidine unit into a HTS hit compound followed by optimization of the amine linker and the carbamoyl moiety led to the identification of compound 1 as a potential candidate. The identified compound 1 showed high selectivity for M1 and M4 muscarinic acetylcholine receptors with M4 partial agonistic activity. In addition, compound 1 showed good brain penetration and reversed methamphetamine-induced hyperlocomotion in rats (ED50=3.0 mg/kg, sc).

Discovery of N-sulfonyl-7-azaindoline derivatives as potent, orally available and selective M(4) muscarinic acetylcholine receptor agonists.

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M4 muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M4 muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M4 mAChR agonist. Compound 1 showed a highly selective M4 mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1-10 mg/kg, po).

Discovery of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors selective agonists.

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M1 and M4 muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M1 and M4 mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M(1) and M(4) muscarinic acetylcholine receptors agonists.

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M1 and M4 agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.

Risperidone on apomorphine-induced stereotyped behavior and auditory sensory gating in rhesus monkeys.

The ameliorating effect of risperidone on apomorphine-induced stereotyped behavior and inhibition of auditory sensory gating was investigated using rhesus monkeys. The total duration of the stereotyped behavior observed in the control group was 43.7 ± 23.0 s (n = 3) between 10 and 25 min after vehicle administration, whereas the duration in the apomorphine-treated (0.1 or 0.15 mg/kg i.m., n = 3) group was observed to be significantly prolonged to 413.0 ± 150.6 s. Administration of 0.01, 0.03, 0.1 mg/kg of risperidone 60 min before apomorphine, significantly reduced the duration of this apomorphine-induced stereotyped behavior to 327 ± 104.9 s (n = 3), 8.3 ± 4.2 s (n = 3), and 0.0 ± × 0.0 s (n = 3, t-test: p < 0.05), respectively. Next, the auditory sensory gating test/conditioning (T/C) ratio was used as a bio-marker. The T/C ratio was 0.598 ± 0.0802 in the vehicle-administered control group (n = 4) and was significantly increased to 2.098 ± 0.254 (n = 4) by apomorphine (0.15 mg/kg, i.m.). Administering of risperidone (0.1 mg/kg, s.c.) 30 min before apomorphine treatment significantly restricted the T/C ratio to 0.571 ± 0.0886 (n = 4), compared to the T/C ratio in the vehicle-administered control group. The above results demonstrate, not only behaviorally but also electrophysiologically, the ameliorating effect of risperidone on the induction of schizophrenia-like symptoms by apomorphine in non-human primates.

A novel phosphodiesterase 1 inhibitor reverses L-dopa-induced dyskinesia, but not motivation deficits, in monkeys.

The enzyme phosphodiesterase 1 (PDE1) is highly expressed in the striatum and cortex. However, its role in corticostriatal function has not been fully investigated. The present study was aimed at evaluating the therapeutic potential of PDE1 inhibitors in treating motivation deficits and 3,4-dihydroxy-L-phenylalanine (L-dopa)-induced dyskinesia, which are pathological conditions of the corticostriatal system. We used a novel PDE1 inhibitor 3-ethyl-2-{[trans-4-(methoxymethyl)cyclohexyl]oxy}-7-(tetrahydro-2H-pyran-4-yl)-imidazo[5,1-f][1,2,4]triazin-4(3H)-one (DSR-143136), which was identified in our drug discovery program. Motivation in monkeys was measured using a progressive ratio task. L-Dopa-induced dyskinesia and disability scores were measured in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. DSR-143136 had a high selectivity for PDE1 over other PDE families and 67 other biologic targets. A dopamine D1 receptor antagonist SCH-39166 at 0.01, 0.03 and 0.1 mg/kg potently decreased motivation in monkeys. However, DSR-143136 at 0.3 and 3 mg/kg did not affect motivation deficits induced by low-dose SCH-39166 (0.01 mg/kg). On the other hand, DSR-143136 at 3 mg/kg potently decreased L-dopa-induced dyskinesia in the Parkinsonian monkey model. Importantly, this antidyskinesic efficacy was NOT accompanied by detrimental effects on motor function. Further, this compound decreased on-time with marked or severe dyskinesia, without affecting on-time itself. These findings suggest that PDE1 inhibitor could be a therapeutic candidate for treating L-dopa-induced dyskinesia in Parkinson's disease, but not for motivation deficits.

An eye tracking system for monitoring face scanning patterns reveals the enhancing effect of oxytocin on eye contact in common marmosets.

Eye tracking systems are used to investigate eyes position and gaze patterns presumed as eye contact in humans. Eye contact is a useful biomarker of social communication and known to be deficient in patients with autism spectrum disorders (ASDs). Interestingly, the same eye tracking systems have been used to directly compare face scanning patterns in some non-human primates to those in human. Thus, eye tracking is expected to be a useful translational technique for investigating not only social attention and visual interest, but also the effects of psychiatric drugs, such as oxytocin, a neuropeptide that regulates social behavior. In this study, we report on a newly established method for eye tracking in common marmosets as unique New World primates that, like humans, use eye contact as a mean of communication. Our investigation was aimed at characterizing these primates face scanning patterns and evaluating the effects of oxytocin on their eye contact behavior. We found that normal common marmosets spend more time viewing the eyes region in common marmoset's picture than the mouth region or a scrambled picture. In oxytocin experiment, the change in eyes/face ratio was significantly greater in the oxytocin group than in the vehicle group. Moreover, oxytocin-induced increase in the change in eyes/face ratio was completely blocked by the oxytocin receptor antagonist L-368,899. These results indicate that eye tracking in common marmosets may be useful for evaluating drug candidates targeting psychiatric conditions, especially ASDs.

The atypical antipsychotic blonanserin reverses (+)-PD-128907- and ketamine-induced deficit in executive function in common marmosets.

Antagonism of the dopamine D3 receptor is considered a promising strategy for the treatment of cognitive impairment associated with schizophrenia. We have previously reported that the atypical antipsychotic blonanserin, a dopamine D2/D3 and serotonin 5-HT2A receptor antagonist, highly occupies dopamine D3 receptors at its antipsychotic dose range in rats. In the present study, we evaluated the effects of blonanserin on executive function in common marmosets using the object retrieval with detour (ORD) task. The dopamine D3 receptor-preferring agonist (+)-PD-128907 at 1mg/kg decreased success rate in the difficult trial, but not in the easy trial. Since the difference between the two trials is only cognitive demand, our findings indicate that excess activation of dopamine D3 receptors impairs executive function in common marmosets. Blonanserin at 0.1mg/kg reversed the decrease in success rate induced by (+)-PD-128907 in the difficult trial. This finding indicates that blonanserin has beneficial effect on executive function deficit induced by activation of the dopamine D3 receptor in common marmosets. Next, and based on the glutamatergic hypothesis of schizophrenia, the common marmosets were treated with the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine. Ketamine at sub-anesthetic doses decreased success rate in the difficult trial, but not in the easy trial. Blonanserin at 0.1mg/kg reversed the decrease in success rate induced by ketamine in the difficult trial. The findings of this study suggest that blonanserin might have beneficial effect on executive dysfunction in patients with schizophrenia.

The dopamine D1 receptor agonist SKF-82958 effectively increases eye blinking count in common marmosets.

Eye blinking is a spontaneous behavior observed in all mammals, and has been used as a well-established clinical indicator for dopamine production in neuropsychiatric disorders, including Parkinson's disease and Tourette syndrome [1,2]. Pharmacological studies in humans and non-human primates have shown that dopamine agonists/antagonists increase/decrease eye blinking rate. Common marmosets (Callithrix jacchus) have recently attracted a great deal of attention as suitable experimental animals in the psychoneurological field due to their more developed prefrontal cortex than rodents, easy handling compare to other non-human primates, and requirement for small amounts of test drugs. In this study, we evaluated the effects of dopamine D1-4 receptors agonists on eye blinking in common marmosets. Our results show that the dopamine D1 receptor agonist SKF-82958 and the non-selective dopamine receptor agonist apomorphine significantly increased common marmosets eye blinking count, whereas the dopamine D2 agonist (+)-PHNO and the dopamine D3 receptor agonist (+)-PD-128907 produced somnolence in common marmosets resulting in a decrease in eye blinking count. The dopamine D4 receptor agonists PD-168077 and A-41297 had no effect on common marmosets' eye blinking count. Finally, the dopamine D1 receptor antagonist SCH 39166 completely blocked apomorphine-induced increase in eye blinking count. These results indicate that eye blinking in common marmosets may be a useful tool for in vivo screening of novel dopamine D1 receptor agonists as antipsychotics.

The serotonin 5-HT1A receptor agonist tandospirone improves executive function in common marmosets.

Previous pilot clinical studies have shown that the serotonin 5-HT1A receptor agonist tandospirone has beneficial effect on cognitive deficits associated with schizophrenia. In the present study, we evaluated the cognitive efficacy of tandospirone, given alone or in combination with the antipsychotic blonanserin, risperidone or haloperidol, on executive function in marmosets using the object retrieval with detour (ORD) task. Treatment with tandospirone alone at 20 and 40 mg/kg increased the number of correct responses in the difficult trial, while risperidone (0.3mg/kg) and haloperidol (0.3mg/kg) decreased the number of correct responses in this trial. On the other hand, blonanserin (0.1-0.3mg/kg), an atypical antipsychotic highly selective for dopamine D2/D3 and serotonin 5-HT2A receptors, did not affect the number of correct responses in both the easy and difficult trials. Co-treatment with tandospirone (20mg/kg) and risperidone (0.1-0.3mg/kg) or haloperidol (0.1-0.3mg/kg) did not improve animals' performance in the difficult trial. However, co-treatment with tandospirone and blonanserin (0.1-0.3mg/kg) increased the number of correct responses in the difficult trial. In addition, treatment with the dopamine D1 receptor agonist SKF-81297 at 1mg/kg increased marmosets correct responses in the difficult trial. These results suggest that tandospirone is a promising candidate for the treatment of cognitive deficits associated with schizophrenia and that adjunctive treatment with tandospirone and blonanserin is more appropriate for cognitive deficits than combination therapy with tandospirone and risperidone or haloperidol. The results of this study also indicate that the putative mechanism of action of tandospirone might be related to enhancement of dopamine neurotransmission via activation of the 5-HT1A receptor.

Lack of dopamine D4 receptor affinity contributes to the procognitive effect of lurasidone.

We previously demonstrated among several antipsychotics exhibiting potent dopamine D2 receptor antagonism that only lurasidone, (1R,2S,3R,4S)-N-[(1R,2R)-2-[4-(1,2-benzisothiazol-3-yl)-1-piperazinylmethyl]-1-cyclohexylmethyl]-2,3-bicyclo[2.2.1] heptanedicarboximide hydrochloride, improved performance in the object retrieval detour (ORD) task by marmosets. The mechanisms by which only lurasidone causes enhancements in cognitive function have not yet been established; however, most antipsychotics, except for lurasidone, have been shown to exhibit potent antagonistic activity against the dopamine D4 receptor. The objectives of this study were to evaluate the role of the dopamine D4 receptor on executive function with the selective agonist, Ro10-5824 and antagonist, L-745,870, and elucidate a possible mechanism for the procognitive effect of lurasidone. The effects of these drugs were evaluated in naïve marmosets using the ORD task. Changes in the success rate during the difficult trial in the task were used to assess the cognitive effect of the drugs. Ro10-5824 (0.3-3 mg/kg) increased the success rate in the difficult trial, potentiated the effect of lurasidone, and reversed the cognitive impairment induced by clozapine. Interestingly, the co-administration of L-745,870 with lurasidone decreased the success rate in the difficult trial, whereas the single administration of L-745,870 had no effect. These results suggest that activation of the dopamine D4 receptor may improve executive function, whereas concomitant blockade of dopamine D4 and D2 receptors may have the opposite effect. In addition to the other unique binding profiles of other monoamine receptors, the lack of affinity for the dopamine D4 receptor by lurasidone could also contribute, at least partly, to its cognitive-enhancing effect.

Effects of lurasidone on ketamine-induced joint visual attention dysfunction as a possible disease model of autism spectrum disorders in common marmosets.

Infants with autism have difficulties performing joint visual attention (JVA), defined as following another person's pointing gesture and gaze. Some non-human primates (NHPs) can also perform JVA. Most preclinical research on autism spectrum disorders (ASD) has used rodents as animal models of this social interaction disorder. However, models using rodents fail to capture the complexity of social interactions that are disrupted in ASD. Therefore, JVA impairment in NHPs might be a more useful model of ASD. The aim of this study was to develop an appropriate and convenient ASD model with common marmosets. We first tested whether marmosets were capable of performing JVA. Subsequently, we administered ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, to induce JVA impairment and investigated the effects of lurasidone, a newer antipsychotic agent, on the JVA impairments. An apparatus was constructed using 4 white boxes, which were attached to the corners of a frame. All boxes had a hinged door, and marmosets could easily obtain a reward by pushing the door. An experimenter pointed and gazed at the boxes to inform the marmosets which box contained the reward. Their behavior was scored according to the number of incorrect choices. The JVA score was significantly higher in the cued vs. uncued tasks. Ketamine significantly decreased the JVA score, but lurasidone significantly reversed this effect. These findings suggest that this experimental system could be a useful animal model of neuropsychiatric disorders characterized by NMDA-receptor signaling, including ASD, and that lurasidone might be effective for some aspects of ASD.

Effects of lurasidone on executive function in common marmosets.

Cognitive impairment is one of the major symptoms of schizophrenia, and is considered largely due to dysfunctions in the prefrontal cortex (PFC). Lurasidone, a novel atypical antipsychotic agent with high binding affinity for dopamine D2, serotonin 5-HT7, 5-HT2A and 5-HT1A receptors has been reported to have superior efficacy in rodents' models of cognitive impairment. However, the beneficial effect of lurasidone on cognitive impairment has not been evaluated in non-human primates. In this study, we investigated the effect of lurasidone on executive function, which is one of the cognitive domains, in common marmosets and compared the results to those of other antipsychotics. The effects of lurasidone, haloperidol, olanzapine, risperidone, quetiapine and clozapine on executive function were evaluated in naïve marmosets using the object retrieval with detours (ORD) task. Before drug treatment, marmosets' success rates in the easy trial of the test were almost 90%. However, maximum success in the difficult trial of the task reached only 50% after 8 days of training. Haloperidol, olanzapine and risperidone decreased correct performance even in the easy trial of the task. All drugs, except lurasidone, impaired success rate in the difficult trial. On the other hand, lurasidone dose-dependently increased marmosets' success rates in the difficult trial with significant effect at 10mg/kg. In conclusion, we have shown in this study that lurasidone, unlike conventional antipsychotics, improves cognition associated with executive function in common marmosets. These findings suggest that lurasidone would be more useful for treatment of schizophrenia cognitive impairment than other antipsychotics.

Effects of a dopamine D1 agonist on ketamine-induced spatial working memory dysfunction in common marmosets.

It is considered that functional deficiency of the NMDA receptors in the prefrontal cortex (PFC) is one of the causes of the cognitive impairment observed in schizophrenia. As non-human primates display more developed PFC than rodents, they are considered to be useful experimental animals for improving the predictive validity of models used to discover new drugs for treating cognitive dysfunction. The aim of this study was to develop a convenient model of the cognitive impairment observed in schizophrenia using common marmosets and the CANTAB system and to test whether a full agonist of the dopamine D1 receptor (SKF-81297) was effective against the cognitive impairment induced in this model. We administered the NMDA receptor antagonist ketamine (1.5-16mg/kg, i.m.) to the marmosets to induce cognitive impairment and then evaluated their working memory function using the CANTAB spatial working memory (SWM) test. The marmosets' working memory was impaired by subanesthetic doses of ketamine. Next, we tested the effect of SKF-81297 (3 or 10mg/kg, p.o.) on this ketamine-induced cognitive dysfunction. The marmosets were administered SKF-81297 30min before the ketamine injection. Pretreatment with SKF-81297 reversed the ketamine-induced cognitive deficiency. In this study, we found that a D1 receptor agonist, which has been reported to enhance cognitive function, reversed ketamine-induced cognitive impairment in marmosets, which suggests that our marmoset model could be a useful tool for predicting the clinical efficacy of cognitive-enhancing drugs.

Activation of ß-adrenoceptors in the bed nucleus of the stria terminalis induces food intake reduction and anxiety-like behaviors.

We previously demonstrated the critical role of noradrenergic transmission within the ventral part of the bed nucleus of the stria terminalis (vBNST) in pain-induced aversion. We showed that activation of ß-adrenoceptors in this brain region by intra-vBNST injection of isoproterenol, a ß-adrenoceptor agonist, produced aversive responses. In the present study, we examined the effects of a ß-adrenoceptor agonist injected into the vBNST on food intake and anxiety-like behaviors in male Sprague-Dawley rats. Bilateral intra-vBNST injection of isoproterenol (3 and 10 nmol/side) caused a dose-dependent decrease in food intake; this suppressive effect was reversed by co-administration of timolol, a ß-adrenoceptor antagonist. Dose-dependent (10 and 30 nmol/side) induction of anxiety-like behaviors by isoproterenol was observed in the elevated plus maze (EPM) test, which was also reversed by co-administration of timolol. Off-site control injections of isoproterenol into the lateral ventricle did not show any significant effect in the food consumption and EPM tests. These results suggest that the vBNST is one of the neuroanatomical substrates which may be involved in the close relationship between negative affective states and reduction of food intake, and that noradrenergic transmission within this brain region plays a critical role in inducing anxiety-like behaviors and reduced food intake.

Discovery of novel N-substituted oxindoles as selective m1 and m4 muscarinic acetylcholine receptors partial agonists.

Activation of the M1 and M4 muscarinic acetylcholine receptors is thought to play an important role in improving the symptoms of schizophrenia. However, discovery of selective agonists for these receptors has been a challenge, considering the high sequence homology and conservation of the orthosteric acetylcholine binding site among muscarinic acetylcholine receptor subtypes. We report in this study the discovery of novel N-substituted oxindoles as potent muscarinic acetylcholine receptor partial agonists selective for M1 and M4 over M2, M3, and M5. Among these oxindoles, compound 1 showed high selectivity for the M1 and M4 receptors with remarkable penetration into the central nervous system. Compound 1 reversed methamphetamine- and apomorphine-induced psychosis-like behaviors with low potency to extrapyramidical and peripheral side effects.